Leinamycin (LNM), migrastatin (MGS), dorrigocin (DGN), and lactimidomycin (LTM) encompass a broad spectrum of biological activities. This application proposes (1) to study the biochemistry and genetics of LNM, MGS/DGN, and LTM production as models for "AT-less" type I polyketide synthase (PKS)- catalyzed polyketide biosynthesis, (2) to develop new combinatorial biosynthesis strategies for engineering polyketide structural diversity, and (3) to manipulate LNM, MGS/DGN, and LTM biosyntheses for the production of biologically active compounds. The hypotheses are: (1) "AT-less" type I PKSs represent an unprecedented PKS structure, the studies of which will reveal new insights into the molecular mechanism of PKS catalysis; (2) "AT-less" type I PKS provides new opportunities for PKS engineering, methods and strategies based on which will further expand the number and diversity of polyketide products accessibly by combinatorial biosynthesis; (3) LNM, MGS/DGN, and LTM biosyntheses are unprecedented, the characterization of which will uncover new chemistry for polyketide biosynthesis; and (4) LNM, MGS/DGN, and LTM are excellent starting molecular scaffolds for combinatorial biosynthesis, derivation of which could lead to the discovery of therapeutically useful agents. The outcome of the proposed studies will (1) expand the repertoire of PKS genes for combinatorial biosynthesis; (2) provide new strategies and methods for combinatorial biosynthesis employing "AT-less" type I PKS; and (3) potentially lead to the production of LNM, MGS/DGN, and LTM analogs as novel therapeutic agents. The specific aims for this grant period are: (1) functional analysis of the Inm biosynthetic gene cluster by targeted gene inactivation in vivo in S. atroolivaceus and by biochemical characterization of the LNM hybrid nonribosomal peptide synthetase (NRPS)-"AT-less" type I PKS megasynthetase in vitro; (2) functional analysis of the mgs/dgn biosynthetic gene cluster by targeted gene inactivation in vivo in S. platensis and by biochemical characterization of the MGS/DGN "AT-less" type I PKS in vitro; (3) functional analysis of the Itm biosynthetic gene cluster by targeted gene inactivation in vivo in S. amphibiosporus and by biochemical characterization of the LTM "AT-less" type I PKS in vitro; (4) development of combinatorial biosynthesis strategies and methods based on the LNM, MGS/DGN, LTM biosynthetic machinery for novel polyketide production.